HIV infection is characterized by high rates of viral turnover throughout the disease process, eventually leading to CD4 depletion and disease progression. Wei X, Ghosh S K, Taylor M E, et al. (1995) Nature 343, 117-122 and Ho D D, Naumann A U, Perelson A S, et al. (1995) Nature 373, 123-126. The aim of antiretroviral therapy is to achieve substantial and prolonged suppression of viral replication. Achieving sustained viral control is likely to involve the use of sequential therapies, generally each therapy comprising combinations of three or more antiretroviral drugs. Choice of initial and subsequent therapy should, therefore, be made on a rational basis, with knowledge of resistance and cross-resistance patterns being vital to guiding those decisions. The primary rationale of combination therapy relates to synergistic or additive activity to achieve greater inhibition of viral replication. The tolerability of drug regimens will remain critical, however, as therapy will need to be maintained over many years.
In an untreated patient, some 1010 new viral particles are produced per day. Coupled with the failure of HIV reverse transcriptase (RT) to correct transcription errors by exonucleolytic proofreading, this high level of viral turnover results in 104 to 1010 mutations per day at each position in the HIV genome. The result is the rapid establishment of extensive genotypic variation. While some template positions or base pair substitutions may be more error prone (Mansky L M, Temin H M (1995) J Virol 69, 5087-5094) (Schinazi R F, Lloyd R M, Ramanathan C S, et al. (1994) Antimicrob Agents Chemother 38, 268-274), mathematical modeling suggests that, at every possible single point, mutation may occur up to 10,000 times per day in infected individuals.
For antiretroviral drug resistance to occur, the target enzyme must be modified while preserving its function in the presence of the inhibitor. Point mutations leading to an amino acid substitution may result in change in shape, size or charge of the active site, substrate binding site or surrounding regions of the enzyme. Mutants resistant to antiretroviral agents have been detected at low levels before the initiation of therapy. (Mohri H, Singh M K, Ching W T W, et al. (1993) Proc Natl Acad Sci USA 90, 25-29) (Nájera I, Richman D D, Olivares I, et al. (1994) AIDS Res Hum Retroviruses 10, 1479-1488) (Nájera I, Holguin A, Quiñones-Mateu E, et al. (1995) J Virol 69, 23-31). However, these mutant strains represent only a small proportion of the total viral load and may have a replication or competitive disadvantage compared with wild-type virus. (Coffin J M (1995) Science 267, 483-489). The selective pressure of antiretroviral therapy provides these drug-resistant mutants with a competitive advantage and thus they come to represent the dominant quasispecies (Frost S D W, McLean A R (1994) AIDS 8, 323-332) (Kellam P, Boucher C A B, Tijnagal J M G H (1994) J Gen Virol 75, 341-351) ultimately leading to drug resistance and virologic failure in the patient.
Non-Nucleoside Reverse Transcriptase Inhibitors
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are a chemically diverse group of compounds which are potent inhibitors of HIV-1 RT in vitro. These compounds include pyridinone derivatives, bis(heteroaryl)piperazines (BHAPs) such as delavirdine and atevirdine, the dipyridodiazepinone nevirapine, the thymine derivative groups TSAO and HEPT, an α-anilino phenylacetamides (α-APA) compound loviride, and the quinoxaline-class inhibitors such as (HBY-097), the benzodiazepin-one and thione (TIBO) compounds and the pyridinone derivatives (L-697,661). For overviews see (DeClercq E. (1996) Rev Med Virol 6, 97-117) (Emini E A (1996) Antiviral Drug Resistance, ed. D D Richman, John Wiley & Sons, Ltd. High-level resistance to individual compounds appears to develop rapidly, often within a few weeks of initiating monotherapy, frequently involving only single-point mutations and in many cases leading to considerable cross-resistance to other NNRTIs. Most mutations reported occur in the codon groups 100-108 and 181-190 which encode for the two β-sheets adjacent to the catalytic site of the RT enzyme (Kohlstaedt L A, Wang J, Friedman J M, et al. (1992) Science 256, 1783-90). The NNRTI binding pocket, as it has been described, is a hydrophobic non-substrate binding region of RT where these agents directly interact with RT. They inhibit activity by interfering with mobility of the ‘thumb’ subdomain, or disrupting the orientation of conserved aspartic acid side chains essential for catalytic activity (D'Aquilla R T. (1994) Clin Lab Med 14, 393-423) (Arnold E., Ding J., Hughes S H, et al. (1995) Curr Opin Struct Biol 5, 27-38).
Mutations conferring reduced susceptibility to nevirapine have been described at codons 98, 100, 103, 106, 108, 181, 188 and 190 (Richman D D, Havlir D, Corbeil J. (1994) J Virol 68, 1660-1666). The most frequently selected variant during nevirapine monotherapy is a Tyr181_Cys (Y181C) mutation which results in a 100-fold reduction in sensitivity to this agent, with reduced susceptibility to the pyridinone derivatives L-696,229 and L-697,661 (Arnold, Ibid). TSAO also has limited activity in the presence of the 181 mutation, but maintains activity in the presence of mutations at codons 100 and 103 and in vitro selects for a unique mutation, GLU138_Lys (E138K), in the region where it most closely interacts with RT (Richman D D, Ibid) (Richman D D, Shih C-K, Lowy I, et al. (1991) Proc Natl Acad Sci USA 88, 11241-11245).
Resistance to loviride when used as monotherapy develops in most patients by week 24. It has been mapped to a range of codons 100-110; 181-190), most commonly codon 103 (Staszewski S, Miller V, Kober A, et al. (1996) Antiviral Ther 1, 42-50). During combination therapy using loviride with zidovudine or zidovudine plus lamivudine, variants at codons 98 and 103 were the most frequent mutations detected at 24 weeks (Staszewski S, Miller V, Rehmet S, et al. (1996) AIDS 10, F1-7).
Although the 101, 103 and 181 mutations also confer cross-resistance to BHAPs. (Balzarini J, Karlsson A, Pérez-Pérez M-J, et al. (1992) Virology 192, 246-253) the characteristic P236L substitution selected for by these agents in vitro appears to sensitize RT to some other NNRTIs, reducing the IC50 for nevirapine, for example, 7- to 10-fold, without influencing sensitivity to nucleoside analogues (Staszewski S, Ibid). This mutation at codon 236 has not been observed in clinical isolates during atevirdine therapy, although other resistance-conferring mutations at codons 103 and 181 have been reported during monotherapy as well as at codons 101, 188, 233 and 238 during combination therapy with zidovudine.
While HBY-097 may initially select for a mutation at codons 190 in vitro, further passage consistently selects for mutations at RT codon 74 and 75, with some mutant viruses showing decreased sensitivity to didanosine and stavudine, but not zidovudine (Kleim J-P, Rösner M, Winkler I, et al. (1995) J Acquir Immune Defic Syndr Suppl 3, 2).
Mutation at codon 181 has been reported to antagonize zidovudine resistance due to the typical 41 and 215 codon mutations, Zhang D, Caliendo A M, Eron J J, et al. (1994) Antimicrob Agents Chemother 38, 282-287) suggesting that combination therapy with some NNRTIs and zidovudine may be feasible. Although an HIV mutant with triple resistance to zidovudine, didanosine and nevirapine has been described in vitro, (Larder B A, Kellam P, Kemp S D (1993) Nature 365, 451-453) treatment with this triple combination does provide superior immunological and virological responses to treatment with zidovudine plus didanosine alone over a 48-week period in patients with CD4 cell counts <350/mm.
Combination therapy with zidovudine and the pyridinone derivative L-697,661 prevents the appearance of the codon 181 mutation typically selected during monotherapy with this NNRTI, delaying the appearance of high-level resistance to this compound. Changes in susceptibility to zidovudine were not examined in this study. (Staszewski S, Massari F E, Kober A, et al. (1995) J Infect Dis 171, 1159-1165). Concomitant or alternating zidovudine therapy does not delay the appearance of resistance during nevirapine therapy; (Richman D D, Ibid) (Nunberg J H, Schleif W A, Boots E J, et al. (1990) J Virol 65, 4887-4892) (DeJong M D, Loewenthl M, Boucher C A B, et al. (1994) J Infect Dis 169, 1346-1350) (Cheeseman S H, Havlir D, McLaughlin M M, et al. (1995) J Acquir Immune Defic Syndr 8, 141-151) however, the 181 mutant is not being observed during combination, the most common change being at codon 190 (Richman D D, Ibid). This suggests that the codon 181 mutation which is antagonistic to zidovudine resistance in vitro is not compatible, or not preferred in vivo, selection favoring other mutations which allow for reduced susceptibility to this NNRTI concomitant with zidovudine resistance.
The rapid development of reduced susceptibility to the NNRTIs suggests limited utility of these agents, particularly as monotherapies, and has led to the modification of these molecules in an attempt to delay the appearance of drug-resistant virus. A ‘second generation’ NNRTI, the pyridinone derivative L-702,019, demonstrated only a 3-fold change in IC between wild-type and codon 181 mutant HIV-1, and required multiple mutations to engender high-level resistance (Goldman M E, O'Brien J A, Ruffing T L, et al. (1993) Antimicrob Agents Chemother 37, 947-949).
It is an object of this invention to provide a drug susceptibility and resistance test capable of showing whether a viral population in a patient is resistant to a given prescribed drug. Another object of this invention is to provide a test that will enable the physician to substitute one or more drugs in a therapeutic regimen for a patient that has become resistant to a given drug or drugs after a course of therapy. Yet another object of this invention is to provide a test that will enable selection of an effective drug regimen for the treatment of HIV infections and/or AIDS. Yet another object of this invention is to provide the means for identifying the drugs to which a patient has become resistant, in particular identifying resistance to non-nucleoside reverse transcriptase inhibitors. Still another object of this invention is to provide a test and methods for evaluating the biological effectiveness of candidate drug compounds which act on specific viruses, viral genes and/or viral proteins particularly with respect to viral drug resistance associated with non-nucleoside reverse transcriptase inhibitors. It is also an object of this invention to provide the means and compositions for evaluating HIV antiretroviral drug resistance and susceptibility. This and other objects of this invention will be apparent from the specification as a whole.